Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of inciting antigens, including viruses, chemicals, and drugs. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its evolution, sometimes years after normalization of blood counts, to other hematologic diseases such as paroxysmal nocturnal hemoglobinuria (PNH), which derive from clones of hematopoietic stem cells. Our laboratory studies have focused on the immune pathophysiology of AA, identification of a viral antigen, and the mechanism of late clonal evolution. Studies of etiology have continued to focus on an unknown hepatitis virus in the post-hepatitis AA syndrome (see Z01 HL 02319-14 HB). Current studies of the immune system's role in bone marrow suppression have focused on gamma-interferon, a lymphokine that inhibits hematopoiesis in vitro and in vivo. We have established an animal model for both AA and transfusion-associated graft-versus-host disease. A congenic mouse strain is employed; lymph node cells from an F1 hybrid are injected into the parental strain, where they rapidly induce bone marrow destruction due to major histocompatibility antigen differences. In this model, both helper and cytotoxic lymphocytes are required, gamma-interferon is produced, and bone marrow destruction is severe. We have established that anti-lymphocyte globulin (ATG) as well as cyclosporine ameliorate disease; anti-gamma-interferon monoclonal antibodies also prevent death and severe blood count depression, establishing for the first time an effector role for this lymphokine in immune marrow destruction. We have measured gamma-interferon in circulating peripheral blood cells in patients with AA using flow cytometry; the presence of these cells appears to correlate with the rapidity and extent of response to immunosuppressive therapy. Intracellular cytokine measurements are also the basis for TH1/TH2 and TC1/TC2 shifts as a result of in vitro treatments. While ATG does not appear to immunomodulate the character of the immune response, cyclosporine, various androgen preparations, and growth factor combinations in vitro and, in the latter case, also in vivo in normal volunteers after the TH1/TH2 immune response in favor of the TH2 component. These results are suggestive of a novel mechanism of action for these various therapeutic agents. Studies of hematopoiesis in vitro have shown that in inhibition of apoptosis by blockade of the caspase pathway is involved in lymphocyte destruction in acquired immunodeficiency syndrome, and ICE blockade in vitro may be useful to expand primitive hematopoietic stem cell numbers in tissue culture. In studies of late clonal disease following successful treatment of AA, we have focused on PNH. Previously, we established that absence of glycophosphoinositol-anchored proteins conferred marked resistance on natural killer cell cytolysis of a lymphoid cell line target; these experiments have been expanded to a hematopoietic target, paired K562 cells that lack or express glycophosphoinositol-anchored proteins. Current studies are directed to identification of the precise protein(s) involved in this interaction. We also have developed a sensitive and simple method for the detection of glycophosphoinositol-anchored proteins on hematopoietic cells from patient material, utilizing two color flow cytometry of polymorphonuclear cells. In studies of patient material we have established that approximately 20% of patients with AA present with evidence of a PNH clone and that this number is stable for several years. In addition, PNH clones are not observed in non-immune forms of bone marrow failure, such as after bone marrow transplantation or cancer chemotherapy, nor does PNH develop as a result of ATG treatment for renal allograft rejection. However, a proportion of patients with myelodysplasia also show evidence of a PNH clone, and for these cases such cells are highly predictive of response to immunosuppressive therapy. Our results are consistent with the hypothesis that PNH represents an escape mechanism in immune-mediated bone marrow failure, and that two steps are required for the development of PNH. Finally, our active clinical program continues. In the area of AA, we have initiated a randomized, cross-over design trial to compare ATG with high dose cyclophosphamide. Approximately 15 patients have been entered into this protocol with hematologic responses observed in about 70% in each arm. Long-term evaluation and larger numbers of patients will be required to determine whether cytotoxic chemotherapy can prevent relapse and the late evolution to clonal hematologic diseases.